DOCSLIB.ORG

The Metabolism of Sepiapterin in Drosophila Melanogaster; Emphasizing Its Tetrahydro-Form's

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Metabolism of Sepiapterin in Drosophila Melanogaster; Emphasizing Its Tetrahydro-Form's The Metabolism of Sepiapterin in Drosophila melanogaster; emphasizing its Tetrahydro-form's Toshifumi TAIRA National Institute of Genetics, Misima ReceivedMarch 23, 1961 Introduction Eye-color of the wild-type in Drosophila melanogaster consists of two kinds of pigmentary systems, i.e. the brown and the red. It is a well-known fact that the brown pigment is biosynthesized from a tryptophan metabolite, such as 3-hydroxy- kynurenine (Butenandt et al. 1958). Lederer (1940) gave the name " drosopterin " first to the red eye-pigment. During the past few years, the evidences have been accumulated that the red eye-pigment of this insect is a pteridine derivative. How- ever, any chemical structure of the red eye-pigment has not been exactly clarified yet. Another yellow eye-pigment, which is a pteridine derivative, has been found in mutant se eyes. Ziegler-Gunder and Hadorn (1958) named "sepiapterin " to the main component of yellow eye-pigment extracted from se eyes. Nawa (1960) has determined its chemical structure to be 2-amino-4-hydroxy-6-lactyl-7, 8-dihydropteridine, although different structures for sepiapterin had been proposed by Forrest and Mitchell (1954), Forrest et al. (1959 a) and Viscontini and Mohlmann (1959) independently. From these investigations, it became clear that those two kinds of eye-pigments are both pteridines. Consequently, the pursuit of process of the pteridine metabolism may have an important role for clarifing the mechanism of the eye-pigment formation. In addition to the two kinds of pteridines mentioned above, there exist some other pteridine derivatives in D. melanogaster, i.e. 2-amino-4-hydroxypteridine (AHP), 2- amino-4-hydroxy-6-(1, 2-dihydroxypropyl)pteridine (biopterin; BP) and 2-amino-4, 7- dihydroxypteridine (isoxanthopterin; IXP) reported by Nawa and Taira (1954), Forrest and Mitchell (1955) and Viscontini et al. (1955). Ziegler-Gunder (1960) has reported on tetrahydrobiopterin glucoside found in Drosophila eyes. As to the metabolic relation- ships among these pteridine derivatives, only the enzymatic conversion of AHP into IXP has been clarified by Forrest et al. (1956), Nawa et al. (1958), Glassman and Mitchell (1959) and Hubby and Forrest (1960). The present author (1960, 1961 a) has reported investigations on the pterine reductase which converts sepiapterin (dihydro-form) into tetrahydro-form . Further- more, he (1961 b) has proposed a possible metabolic pathway of drosophila pteridines based on the chromatographic and genetical analyses. 1) Contributionfrom National Institute of Genetics, Japan. No. 364. THE METABOLISMOF SEPIAPTERININ DROSOPHILAMELANOGASTER 245 The present paper deals with the pteridine metabolism of Drosophila which can,t be presented by a detailed investigation on the enzymatic reduction of sepiapterin. Material and Methods Prepupae of wild-type (Oregon-R) in Drosophila melanogaster were used for the preparation of purified pterine reductase. Sepiapterin and other pteridine derivatives- such as 2-amino-4-hydroxypteridine (AHP), isoxanthopterin (IXP), biopterin (BP) and 2-amino-4-hydroxypteridine-6-carboxylic acid (PCA), were all provided by Dr. Nawa. Triphosphopyridine nucleotide (TPN) was purchased from Nutritional Biochemicals Corporation, U.S.A. Reduced triphosphopyridine nucleotide (TPNH) was prepared by the reduction of TPN with TPN-transhydrogenase partially purified from chicken heart extract, in the presence of citrate and Mg-. Five milliliters of reaction mixture containing 5 mg of TPN, 0.01 M potassium phosphate buffer, pH 6.8, 3.3 mM of MgSO4, 3.3 mM of citrate, and 0.1 ml of chicken heart enzyme solution were incubated at room temperature until the absorption at 340 m,a reached a constant value. After the addition of 0.1 ml of 1 M NaOH, the mixture was immersed in a boiling water bath for 2 minutes and then centrifuged. The final concentration of TPNH in the supernatant was de- termined spectrophotometrically. The enzymatic conversion of sepiapterin into tetrahydro-form, which will be described in detail below, and that of TPNH into TPN were estimated by spectro- photometrical measurement of the reduction of extinction at 410 m,a for the former and that at 340 m,u for the latter. The chemical natures of pteridines obtained from this experiment were determined by comparisons of their Rf values and ultraviolet absorption spectra with those of the synthesized pteridines. Experimental results Purification of pterine reductase: Fresh wild-type prepupae of D. melanogaster were homogenized at 0°C for 15 seconds with an equal weight of cold 0.1 M potas- sium phosphate buffer, pH 6.2. After centrifugation at 0°C, the supernatant was washed several times with cold acetone (-15°C) and dried in vacuo. The water ex- tract of the crude acetone powder was treated with Dowex 1-chloride in an ice bath to remove cofactors. The precipitate at pH 5.0 was centrifuged off. The reductase was further purified by fractionation with cold acetone. The fraction between 40 and 60% acetone containing almost all of the activity was washed twice with cold acetone and dried in vacuo. The re-extract with cold distilled water (10 mg/ml) was centri- fuged and the residue was discarded. The supernatant had some TPNH-oxidizing activity. Therefore, further purification was followed by fractionation with ammonium sulfate. The fraction between 50 and 65% ammonium sulfate in an ice bath was collected by centrifugation and the precipitate was dissolved with cold distilled water- The residue was centrifuged off. The final supernatant was used as an enzyme solu- tion for the experiment. It seemed to be completely free from TPNH-oxidizing and 246 T. TAIRA TPNH-generating enzymes. Properties of pterine reductase: The purified enzyme could catalize the conversion of sepiapterin, which was a dihydropteridine, in the presence of cofactor. It could be Fig. 1. Cofactor requirement on the reaction of pterine reductase. Enzyme solution, 0.3 ml, was incubated in a medium containing 2 x 10-4M cofactors, l x 10-4M sepiapterin and 0.01 M phosphate buffer, pH 6.5. The total volume was 3 ml. P- Fig. 2. Rate of the reaction and pH of the medium . The enzyme solution, 0.3 ml, was incubated for 30 min . in a medium containing 1 X 10-4M sepiapterin, 2 x 10-4M TPNH, and 0.01M phosphate buffer . The total volume was 3 ml. THE METABOLISMOF SEPIAPTERININ PROSOPHILAMELANOGASTER 247 detected photometrically by disappearance of the yellow color and fluorescence charac- teristic of sepiapterin. For the catalytic reaction, the requirement for TPNH as a cofactor was quite specific and could not be replaced by TPN, DPN (diphosphopyridine nucleotide) or DPNH (reduced DPN), as shown in Fig. 1. Optimum rate of the reac- tion occurred at about pH 6.5 (Fig. 2). The reaction velocity in the absence of oxygen was more rapid than that in its presence. A violent aeration inhibited completely the reaction; such phenomenon might be due to the decomposition of sepiapterin and TPNH. An effect of phosphate and citrate buffers on the reaction velocity was tested, but a difference between them was detected scarcely. The purified reductase was unstable at room temperature in the presence of oxygen and its half-life was about one hour. However, the reductase was considerably stable at 0°C in the absence of oxygen and it could be stored for a month. Various inhibitors for the reaction was tested ; 2, 4-dinitrophenol (6.5X10-5M) in- hibited completely, and folate showed about 40% inhibition in a concentration from 1.6 x 10-5 M to 4.3 x 10-' M. Potassium cyanide (3.3 X 10-4 M) inhibited scarcely. Both riboflavin (3.3 x 10-5 M) and flavin mononucleotide (2.0 x 10-4 M) did not affect to the reaction. Cystein (2.3 x 10-6 M) was used as SH-group, but it did not inhibit unex- pectedly. According to the previous reports of the present author (1960, 1961 c), the ex- tract of Hnr3 male has contained a high concentration of inhibitor. This substance was isolated and purified chromatographically. It was stable for a high temperature and it was chromatographically indistinguishable from biopterin (see Table 2). However, differing from biopterin, it was scarcely photo-decomposed by visible light. The purified inhibitor showed 80% inhibition even at the maximum rate. At that time, biopterin showed about 70% inhibition. The crude extract from Hnr3 strain completely inhibited the reaction. Therefore, it has remained to be elucidated whether or not this pteridine is only an actual inhibitor in Hnr3 male." Estimation of product enzymatically reduced from sepiapterin: As mentioned above, the purified enzyme could convert sepiapterin (dihydro-from) in the presence of Table 1. Stoichiometry of pterine reductase* *} The enzyme solution, 0.3 ml, was incubated at 20°C in a medium containing 27.8µM sepiapterin, 45.7µM TPNH and 0.01M citrate buffer, pH 6.5. The total volume was 3 ml. 248 T. TA[RA Wave length ma Fig. 3. Ultraviolet absorption sepctra of sepiapterin, tetrahydrofolate and the reaction product:. ......... sepiapterin at pH 11.0; - - - - sepiapterin at pH 1.0; tetrahydrofolate at pH 1.0; the reaction product from sepiapterin at pH 1.0. TPNH, and that could be detected spectrophotometrically by disappearance of the- yellow color and fluorescence characteristic of sepiapterin. In order to identify what was the actual product from this enzymatic reaction, the reduction of extinction at_ 410 mit for sepiapterin and that at 340 m/c for TPNH were measured stoichiometrical ly. The result was shown in Table 1. Except for the value at 10 minutes of in- cubation time, the molar ratio of sepiapterin to TPNH consumed in this reaction medium was nearly 1.0. As shown in Fig. 3, the curve of the reaction product from sepiapterin was very similar to those of tetrahydropteridines reported by Viscontini and Weilenmann (1958) and Kaufman (1959).
Load more

Recommended publications
  • Folate and Pterin Metabolism by Cancer Cells in Culture1
    [CANCER RESEARCH 38, 2378-2384, August 1978) 0008-5472/78/0038-0000$02.00 Folate and Pterin Metabolism by Cancer Cells in Culture1 Baldassarre Stea, Peter S. Backlund, Jr., Phillip B. Berkey, Arthur K. Cho, Barbara C. Halpern, Richard M. Halpern, and Roberts A. Smith2 Departments of Chemistry (B. S., P. S. B., P. B. B., B. C. H.¡,Pharmacology ¡A.K. C.], Medicine, ¡Ft.M. H.]. and Chemistry [Ft. A. SJ and Molecular Biology Institute ¡R.M. H., R. A. S.], University of California, Los Angeles, California 90024 ABSTRACT cells, a significant peak of radioactivity was observed in the blue-fluorescent region. This peak of radioactivity was ab Malignant cells grown in culture excrete into their sent in chromatograms of growth media of normal cells growth medium a folate catabolite that can be seen as a grown under the same conditions. blue-fluorescent region on paper chromatograms of such We tentatively identified this blue-fluorescent compound media. This folate catabolite has now been identified by as Pt-6-CHO,3 primarily on the basis of its inhibitory power paper chromatography, thin-layer chromatography, and toward xanthine oxidase. However, we later found that Pt- combined gas chromatography-mass spectrometry as 6- 6-CH2OH also is a potent inhibitor of the same enzyme hydroxymethylpterin and not as pterin-6-carboxaldehyde system. This prompted us to identify by unequivocal means as previously reported. the fluorescent folate catabolite characteristic of cultured Moreover, when pterin-6-carboxaldehyde was added to malignant cells. the growth medium of logarithmically growing malignant Here we report the definitive identification of this blue- cells, it was primarily reduced to 6-hydroxymethylpterin.
    [Show full text]
  • Relevance to Neurological Damage
    Postgrad Med J: first published as 10.1136/pgmj.62.724.113 on 1 February 1986. Downloaded from Postgraduate Medical Journal (1986) 62, 113-123 Pteridines and mono-amines: relevance to neurological damage I. Smith, D.W. Howells and K. Hyland Institute ofChildHealth (University ofLondon), 30 Guilford Street, London WCIN2NR, UK. Summary: Patients with phenylalanine hydroxylase deficiency show increased concentrations of biopterins and neopterins, and reduced concentrations ofserotonin and catecholamines, when phenylalan- ine concentrations are raised. The pterin rise reflects increased synthesis of dihydroneopterin and tetrahydrobiopterin, and the amine fall a reduction in amine synthesis due to inhibition by phenylalanine of tyrosine and typtophan transport into neurones. The pterin and amine changes appear to be independent of each other and are present in the central nervous system as well as the periphery; they disappear when phenylalanine concentrations are reduced to normal. Patients with arginase deficiency show a similar amine disturbance but have normal pterin levels. The amine changes probably contribute neurological symptoms but pterin disturbance is not known to affect brain function. Patients with defective biopterin metabolism exhibit severely impaired amine synthesis due to tetrahydrobiopterin deficiency. Pterin concentrations vary with the site of the defect. Symptoms include profound hypokiesis and other features of basal ganglia disease. Neither symptoms nor amine changes are relieved by controlling phenylalanine concentrations. Patients with dihydropteridine reductase (DHPR) deficiency accumulate dihydrobiopterins and develop secondary folate deficiency which resembles that occurring in patients with defective 5,10-methylene tetrahydrofolate reductase activity. The latter disorder is also associated writh Parkinsonisn and defective amine and pterin turnover in the and a occurs in In central nervous system, demyelinating illness both disorders.
    [Show full text]
  • Diosynrhesis of Neopterin, Sepioprerin, Ond Diopterin in Rar Ond Humon Oculor Tissues
    768 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / May 1985 Vol. 26 before and after the addition of beta-glucuronidase.4 ported by the Juvenile Diabetes Foundation and by the University In circumstances where the actual plasma free fluo- of London, Central Research Fund. The HPLC was purchased with an MRC grant to Dr. Michael J. Neal. Submitted for publi- rescein has not been measured, the term "plasma- cation: April 12, 1984. Reprint requests: Dr. P. S. Chahal, Depart- free fluorescence" should be quoted. It is then better ment of Medicine, Hammersmith Hospital, Du Cane Road, London to measure overall fluorescence (fluorescein and the W12 OHS, England. glucuronide metabolite) in protein-free plasma ultra- filtrate and the fluorescence appearing in the ocular References compartments using the same excitor and emission filters. 1. Araie M, Sawa M, Nagataki S, and Mishima S: Aqueous Fluorescein glucuronide is a potential source of humor dynamics in man as studied by oral fluorescein. Jpn J Ophthalmol 24:346, 1980. variability in studies of blood-ocular dynamics using 2. Zeimer RC, Blair NP, and Cunha-Vaz JG: Pharmacokinetic fluorescein. Its exact role has yet to be established. interpretation of vitreous fluorophotometry. Invest Ophthalmol VisSci 24:1374, 1983. Key words: Blood-ocular barriers, diabetes, plasma ultrafil- 3. Chen SC, Nakamura H, and Tamura Z: Studies on metabolite trate, fluorescein glucuronide, fluorescence, protein-binding of fluorescein in rabbit and human urine. Chem Pharmacol Acknowledgments. Technical assistance was given by Dr. Bull 28:1403, 1980. J. Cunningham, Ian Joy, and Margaret Foster. 4. Chen SC, Nakamura H, and Tamura Z: Determination of fluorescein and fluorescein monoglucuronide excreted in urine.
    [Show full text]
  • Isolation and Expression of Rat Liver Sepiapterin Reductase Cdna
    Proc. Nat!. Acad. Sci. USA Vol. 87, pp. 6436-6440, August 1990 Genetics Isolation and expression of rat liver sepiapterin reductase cDNA (tetrahydrobiopterin/biopterin/pyruvoyltetrahydropterin/aromatic amino acid hydroxylase/molecular cloning) BRUCE A. CITRON*, SHELDON MILSTIEN*, JOANNE C. GUTIERREZt, ROBERT A. LEVINE, BRENDA L. YANAK*§, AND SEYMOUR KAUFMAN*¶ *Laboratory of Neurochemistry, Room 3D30, Building 36, and tLaboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; and tLaboratory of Molecular Neurobiology, Lafayette Clinic, 951 East Lafayette, Detroit, MI 48207 Contributed by Seymour Kaufman, June 7, 1990 ABSTRACT Sepiapterin reductase (7,8-dihydrobiopterin: either case, the final carbonyl reduction to form tetrahydro- NADP+ oxidoreductase, EC 1.1.1.153) catalyzes the terminal biopterin must be catalyzed by sepiapterin reductase. step in the biosynthetic pathway for tetrahydrobiopterin, the In the present study, we have screened a rat liver cDNA cofactor necessary for aromatic amino acid hydroxylation. We library with a specific antibody that reacts positively to the report here the isolation of a cDNA clone for rat liver sepia- enzyme band on Western blots (19). These clones were used pterin reductase. The cDNA has been excised from a lambda to further characterize the enzyme and to begin to elucidate vector and the DNA sequence was determined. The insert the functional sites ofthe enzymes in the tetrahydrobiopterin contains the coding sequence for at least 95% of the rat enzyme biosynthetic pathway. 11 and is fused to the Escherichia coli (3-galactosidase N-terminal segment and the lac promoter. The N-terminal region of the clone contains an extraordinarily high G+C content.
    [Show full text]
  • Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels
    biomolecules Article Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels Benjamin Neuditschko 1,2,† , Marlene Leibetseder 1,† , Julia Brunmair 1 , Gerhard Hagn 1 , Lukas Skos 1, Marlene C. Gerner 3 , Samuel M. Meier-Menches 1,2,4 , Iveta Yotova 5 and Christopher Gerner 1,4,* 1 Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Straße 38, 1090 Vienna, Austria; [email protected] (B.N.); [email protected] (M.L.); [email protected] (J.B.); [email protected] (G.H.); [email protected] (L.S.); [email protected] (S.M.M.-M.) 2 Department of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Straße 42, 1090 Vienna, Austria 3 Division of Biomedical Science, University of Applied Sciences, FH Campus Wien, Favoritenstraße 226, 1100 Vienna, Austria; [email protected] 4 Joint Metabolome Facility, Faculty of Chemistry, University of Vienna, Waehringer Straße 38, 1090 Vienna, Austria 5 Department of Obstetrics and Gynaecology, Medical University of Vienna, Spitalgasse 23, 1090 Vienna, Austria; [email protected] Citation: Neuditschko, B.; * Correspondence: [email protected] Leibetseder, M.; Brunmair, J.; Hagn, † Authors contributed equally. G.; Skos, L.; Gerner, M.C.; Meier-Menches, S.M.; Yotova, I.; Abstract: Endometriosis is a benign disease affecting one in ten women of reproductive age world- Gerner, C. Epithelial Cell Line wide. Although the pain level is not correlated to the extent of the disease, it is still one of the Derived from Endometriotic Lesion cardinal symptoms strongly affecting the patients’ quality of life.
    [Show full text]
  • Treatable Inborn Errors of Metabolism Presenting As Cerebral Palsy Mimics
    Leach et al. Orphanet Journal of Rare Diseases 2014, 9:197 http://www.ojrd.com/content/9/1/197 RESEARCH Open Access Treatable inborn errors of metabolism presenting as cerebral palsy mimics: systematic literature review Emma L Leach1,2, Michael Shevell3,4,KristinBowden2, Sylvia Stockler-Ipsiroglu2,5,6 and Clara DM van Karnebeek2,5,6,7,8* Abstract Background: Inborn errors of metabolism (IEMs) have been anecdotally reported in the literature as presenting with features of cerebral palsy (CP) or misdiagnosed as ‘atypical CP’. A significant proportion is amenable to treatment either directly targeting the underlying pathophysiology (often with improvement of symptoms) or with the potential to halt disease progression and prevent/minimize further damage. Methods: We performed a systematic literature review to identify all reports of IEMs presenting with CP-like symptoms before 5 years of age, and selected those for which evidence for effective treatment exists. Results: We identified 54 treatable IEMs reported to mimic CP, belonging to 13 different biochemical categories. A further 13 treatable IEMs were included, which can present with CP-like symptoms according to expert opinion, but for which no reports in the literature were identified. For 26 of these IEMs, a treatment is available that targets the primary underlying pathophysiology (e.g. neurotransmitter supplements), and for the remainder (n = 41) treatment exerts stabilizing/preventative effects (e.g. emergency regimen). The total number of treatments is 50, and evidence varies for the various treatments from Level 1b, c (n = 2); Level 2a, b, c (n = 16); Level 4 (n = 35); to Level 4–5 (n = 6); Level 5 (n = 8).
    [Show full text]
  • GCH1 Gene GTP Cyclohydrolase 1
    GCH1 gene GTP cyclohydrolase 1 Normal Function The GCH1 gene provides instructions for making an enzyme called GTP cyclohydrolase 1. This enzyme is involved in the first of three steps in the production of a molecule called tetrahydrobiopterin (BH4). Other enzymes help carry out the second and third steps in this process. Tetrahydrobiopterin plays a critical role in processing several protein building blocks ( amino acids) in the body. For example, it works with the enzyme phenylalanine hydroxylase to convert an amino acid called phenylalanine into another amino acid, tyrosine. Tetrahydrobiopterin is also involved in reactions that produce chemicals called neurotransmitters, which transmit signals between nerve cells in the brain. Specifically, tetrahydrobiopterin is involved in the production of two neurotransmitters called dopamine and serotonin. Among their many functions, dopamine transmits signals within the brain to produce smooth physical movements, and serotonin regulates mood, emotion, sleep, and appetite. Because it helps enzymes carry out chemical reactions, tetrahydrobiopterin is known as a cofactor. Health Conditions Related to Genetic Changes Dopa-responsive dystonia More than 140 mutations in the GCH1 gene have been found to cause dopa-responsive dystonia. This condition is characterized by a pattern of involuntary muscle contractions ( dystonia), tremors, and other uncontrolled movements and usually responds to treatment with a medication called L-Dopa. Dopa-responsive dystonia results when one copy of the GCH1 gene is mutated in each cell. Most GCH1 gene mutations that cause this condition change single amino acids in the GTP cyclohydrolase 1 enzyme. Researchers believe that the abnormal enzyme may interfere with the activity of the normal version of GTP cyclohydrolase 1 that is produced from the copy of the gene with no mutation.
    [Show full text]
  • Inherited Disorders of Neurotransmitters: Classification and Practical Approaches for Diagnosis and Treatment
    Published online: 2018-10-29 2 Review Article Inherited Disorders of Neurotransmitters: Classification and Practical Approaches for Diagnosis and Treatment Heiko Brennenstuhl1 Sabine Jung-Klawitter1 Birgit Assmann1 Thomas Opladen1 1 Division of Neuropediatrics and Metabolic Medicine, Department of Address for correspondence Prof. Dr. Thomas Opladen, MD, Division General Pediatrics, University Children’s Hospital Heidelberg, of Neuropediatrics and Metabolic Medicine, Department of General Heidelberg, Germany Pediatrics, Im Neuenheimer Feld 430, D-69120 Heidelberg, Germany (e-mail: [email protected]). Neuropediatrics 2019;50:2–14. Abstract Neurotransmitter deficiencies are rare neurological disorders with clinical onset during childhood. The disorders are caused by genetic defects in the enzymes involved in synthesis, degradation, or transport of neurotransmitters or by defects in the cofactor biosynthesis such as tetrahydrobiopterin (BH4). With the newly described DNAJC12 deficiency, a chaperon-associated neurotransmitter disorder, the pathophysiological spectrum has been broadened. All deficiencies result in a lack of monoamine neurotransmitters, especially dopamine and its products, with a subset leading to decreased levels of serotonin. Symptoms can occur already in the neonatal period. Keywords Classical signs are hypotonia, movement disorders, autonomous dysregulations, and ► inherited monoamine impaired development. Diagnosis depends on quantitative detection of neurotrans- neurotransmitter mitters in cerebrospinal
    [Show full text]
  • Optimization of Expression Conditions Enhances Production Of
    J. Microbiol. Biotechnol. (2015), 25(10), 1709–1713 http://dx.doi.org/10.4014/jmb.1506.06034 Research Article Review jmb Optimization of Expression Conditions Enhances Production of Sepiapterin, a Precursor for Tetrahydrobiopterin Biosynthesis, in Recombinant Escherichia coli Eun-Hee Park1, Won-Heong Lee2, Mi-Hee Jang1, and Myoung-Dong Kim1* 1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea 2Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea Received: June 15, 2015 Revised: July 16, 2015 Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a well- Accepted: July 18, 2015 known cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5’-triphosphate cyclohydrolase 1 (GCH1) and human 6- First published online pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The July 22, 2015 optimum conditions for T7 promoter–driven expression of GCH1 and PTPS were 30°C and *Corresponding author 0.1 mM isopropyl-β-D-thioglucopyranoside (IPTG). The maximum sepiapterin concentration Phone: +82-33-250-6458; of 88.1 ± 2.4 mg/l was obtained in a batch cultivation of the recombinant E. coli, corresponding Fax: +82-33-259-5565; to an 18-fold increase in sepiapterin production compared with the control condition (37°C E-mail: [email protected] and 1 mM IPTG). pISSN 1017-7825, eISSN 1738-8872 Copyright© 2015 by Keywords: Sepiapterin, GTP cyclohydrolase 1 (GCH1), 6-pyruvoyltetrahydropterin synthase The Korean Society for Microbiology (PTPS), Escherichia coli and Biotechnology Tetrahydrobiopterin (BH4) is a well-known essential ovarian cancer cells.
    [Show full text]
  • An Ab Initio and Density Functional Theory Study on Neutral Pterin Radicals
    Pteridines 2015; 26(4): 135–142 Gilbert Reibnegger* An ab initio and density functional theory study on neutral pterin radicals DOI 10.1515/pterid-2015-0008 responsible for a remarkably complex chemical behaviour Received May 8, 2015; accepted June 29, 2015; previously published of pteridine itself and also of the many derivatives thereof. online August 7, 2015 A large variety of pteridine derivatives occur in apparently all living organisms, exerting a multitude of different Abstract: The electronic structures of the five radicals biochemical/biological functions that are only partially resulting from homolytic elimination of one of the hydro- understood. In several investigations, pteridine radicals gen atoms from the most stable tautomeric form of neutral have been detected experimentally, or at least pteridine pterin were investigated in gas phase as well as in aque- derivatives were observed to modulate the reactivity of ous solution. Molecular wave functions obtained by den- other free radicals [1–18]. sity functional theory were analysed by quantum theory Recently, several studies were published reporting of atoms in molecules and electron localisation functions on the results of quantum chemical calculations of the (ELF). Spin densities of the radicals as well as electrostatic detailed electronic structures of pterin (2-amino-3H-pteri- potential functions were analysed. Radicals resulting from dine-4-on) and derivatives employing density functional elimination of N-bonded hydrogen atoms are more stable theory (DFT) with reasonably sized basis sets [19–22]. In in comparison with radicals obtained after abstraction of the present investigation, I report on such calculations C-bonded hydrogen atoms. N-centred radicals show strong extended to the five radicals derived from the most stable delocalisation of spin density over both heteroaromatic tautomeric form of neutral pterin by homolytic elimina- rings; in C-centred radicals delocalisation does not occur.
    [Show full text]
  • Identification and Age-Dependance of Pteridines in the Head of Adult Mexican Fruit Fly, Anastrepha Ludens N
    J. insect Phwol. Vol. 42, No. 4, pp 359-366, 1996 CopyrIght C lYY6 Elsewer Science Ltd Pergamon 0022-1910(95)00116-6 Printed III Great Bntaln. All rights reserved 0022-lYlOiY6 $15.00 + 0.00 Identification and Age-Dependance of Pteridines in the Head of Adult Mexican Fruit Fly, Anastrepha ludens N. TOMIC-CARRUTHERS,*t$ D. C. ROBACKER,? R. L. MANGAN? Received 16 May 1995: revised 21 August 1995 Pteridines from head capsules of adult Anastrepha ludens were identified and evaluated as a tool for age determination of flies. Pteridines were identified by a combination of TLC, HPLC and UV and fluorescence spectroscopy. Tentative identifications were obtained for one red pterine, neodrosopterin, and three orange pterines, drosopterin, isodrosopterin and aurodro- sopterin. Five pterines with blue or blue-green fluorescence were found and four were ident- ified: 7,8-dihydrobiopterin; biopterin; pterin; and pterind-carboxylic acid. An additional blue fluorescing non-pteridine compound was identified as kyurenine. Two pterines with yellow fluorescence were identified as sepiapterin and deoxysepiapterin. Titers of sepiapterin and deoxysepiapterin were age dependent based on TLC and HPLC analyses. HPLC demonstrated that only deoxysepiapterin accumulated sufficiently and over a sufficiently long period of adult life to be useful as a quantitative tool for determining age of adult flies. The HPLC method developed in this work specifically for separation of deoxysepiapterin from sepiapterin and other pteridines used reverse-phase (C18) with a mobile phase of 30% methanol in water and detection at 420 nm. Pteridines Sepiapterin Deoxysepiapterin Age Anastrepha ludens INTRODUCTION the main locations of accumulation, at least in adult Fluorescent compounds from insects have been the object insects, are the head and the integument.
    [Show full text]
  • 34Th International Winter Workshop Clinical, Chemical and Biochemical
    DOI 10.1515/pterid-2015-0007 Pteridines 2015; 26(3): 113–133 Abstracts*) 34th International Winter Workshop Clinical, Chemical and Biochemical Aspects of Pteridines and Related Topics Society for Exploitation of Education and Research in Immunology and Infectious Diseases, Innsbruck, Austria in collaboration with The International Society of Pteridinology and The Austrian Society of Laboratory Medicine and Clinical Chemistry Held in Innsbruck, Tyrol, Austria, February 24th–27th, 2015 Scientific committee: Dietmar Fuchs (Innsbruck), Andrea Griesmacher (Innsbruck), Bohuslav Melichar (Olomouc), Gilbert Reibnegger (Graz), Barbara Strasser (Hall), Guenter Weiss (Innsbruck) and Ernst R. Werner (Innsbruck) Organization: Dietmar Fuchs, Sektion für Biologische Chemie, Biozentrum, Medizinische Universität Innsbruck, Innrain 80, 6020 Innsbruck, Austria, e-mail: [email protected] *)These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors’ responsibility. 114 34th International Winter Workshop Circulating neopterin and citrulline concentrations Influence of carbon nanotubes, ZnO and in patients with germ-cell tumors during gold-doped TiO2 nanoparticles on human PBMC chemotherapy in vitro Bartoušková M, Študentová H, Pejpková I, Zezulová M, Adam T, Becker K, Herlin N, Bouhadoun S, Gostner JM, Ueberall F, Schennach Melichar B H, Fuchs D Palacký University Medical School and Teaching Hospital, Olomouc, Divisions of Biological Chemistry and of Medical Biochemistry, Czech Republic Biocenter, Medical University, and Central Institute of Blood ([email protected]) Transfusion and Immunology, University Hospital, Innsbruck, Austria; Au Service des Photons, Atomes et Molécules - Laboratoire Francis Germ-cell tumors are relatively rare neoplasms that affect mostly Perrin, Gif-sur Yvette, France young adults.
    [Show full text]